Nitric oxide synthetase (N)S), the nitric oxide synthesizing enzymes, form a novel important family of proteins. They are implicated in multiple crucial biological functions, particularly in brain. At the present time, two homologous proteins have been cloned and lines of evidence indicate the existence of isoforms. The project I will be involved in is the isolation and the characterization of novel putative nitric oxide genes that are expressed in brain. - Southern and Northern blotting using cloned nitric oxide DNA as probes will\ be carried out for characterizing the best conditions for isolating such isoforms. - Molecular cloning of genes encoding potential NOS isoforms will be conducted using PCR techniques as well as screening of cDNA libraries prepared from discrete regions of the brain. - Localization of the associated transcripts will be determined by in situ hybridization in brain tissues. - Transfection of eukaryotic cells with the candidate genes will be employed to characterize properties of new NOS isoforms.